PROJECT SUMMARY: Bacterial transcription initiation and promoter escape are highly-regulated early steps of gene expression. A critical initiation step occurs when the 5'-end of the nascent RNA clashes with region 3.2 of the promoter specificity factor ?70 (?R3.2) occluding the RNA exit channel. Then, either the occluded channel is cleared to facilitate RNA forward translocation through the RNA exit channel and RNAP to escape the promoter, or the nascent RNA back-translocates into the NTP entry channel, leading to its abortive release. We have recently shown that a fraction of RNAPs get stabilized in a long-lived paused backtracked intermediate during initiation. We have also shown that even after removal of ?R3.2 from the RNA exit channel by the nascent transcript, transcription kinetics is still slower than expected for elongation. Therefore, we hypothesize an additional promoter escape-intermediate further slows down the transition from initiation to elongation, and that both intermediates have regulatory roles. In Aim 1.A, we will elucidate the structures of the transcription initiation complex in these states by using multiple experimentally-derived intramolecular distances as spatial constraints on coarse-grained simulations. In Aim 1.B, we will define the molecular determinants controlling the abundance of these late initiation intermediates. Specifically, we will examine the sequence and order in which ?70 regions are removed from the RNA exit channel during promoter escape for different promoters. In Aim 1.B we hypothesize that: (1) displacement of ?R3 & ?R4 during promoter escape follows a two-step process; (2) the bulge formed in the scrunched DNA template strand of the transcription bubble assists in removal of these ? regions from the RNA exit channel by projecting into the channel. We recently discovered that an excessive number of RNAPs stall at promoters of many genes in vivo that are essential for stress-response and that stalling is enhanced under hyperosmotic conditions in a ?greA/?greB E. coli strain (unpublished). In Aim 2 we will test whether pausing in initiation occurs in live bacteria and serves as a regulatory intermediate for stress response. We will test this hypothesis by high-resolution (1-2 nt) chromosomal DNA mapping & footprinting in vivo techniques. We will also develop in vivo smFRET transcription bubble size assay to test whether pausing in initiation occurs in the bacterial cell through a mechanism similar to that studied in Aim 1. This project will significantly advance the field of transcription for the following reasons: (1) antibiotic resistance is a serious public health concern. Elucidating the mechanisms of bacterial gene regulation is crucial for the development of effective antimicrobial therapy; (2) the conservation of many features of RNAP structure & function from bacteria to humans facilitates modeling of transcription mechanisms for eukaryotic enzymes; (3) the structure of paused-backtracked RNAP in initiation has not yet been determined. Therefore, delineating the spatial rearrangements of ?70 regions blocking the RNA exit channel for different promoters will provide valuable insight into the mechanism of promoter escape.